I. N-linked Oligosaccharide Side Chains
The function of N-linked oligosaccharide side chains that are covalently attached to the majority of eukaryotic secretory and membrane proteins is not yet fully understood. It has been suggested that the N-linked oligosaccharide side chains play a variety of roles including (i) maintenance of the structure and stability of the glycoproteins: (ii) directing the transport of glycoproteins to various subcellular compartments both after biosynthesis and for degradation: and (iii) recognition functions leading to specific adhesion of cells and tissue organization during development or neoplasia (Gibson, R. et al, Trends Biol. Sci., 5:290-293 (1980); Hughes, R. C. et al. Trends Biochem. Sci., 6:228-230 (1981); Pollack, L. et al, J. Cell. Biol., 97:293-300 (1983); Elbein. A. D., Trends Biochem. Sci., 6:219-221 (1981); and Elbein, A. D., Annu. Rev. Biochem., 56:497-534 (1987)).
Studies using drugs that are specific inhibitors of the addition or modification of N-linked oligosaccharide side chains have been performed in attempts to define the functional roles of these carbohydrate moieties (Elbein. A. D., Annu. Rev. Biochem., 56:497-534 (1987)). Further, the advent of recombinant DNA technology has facilitated studies involving the removal of individual glycosylation sites using oligonucleotide mutagenesis (Machamer, C. E. et al, Mol. Cell Biol., 5:3074-3083 (1985); Miyazaki, J. et al, J. Exp. Med., 163:856-871 (1986); Santos-Aguado, J. et al, Molec. and Cell Biol., 7:982-990 (1987); Guan. J. et al, J. Biol. Chem., 263:5306-5313 (1988); Matzuk, M. M. et al, J. Cell Biol., 106:1049-1059 (1988)). The majority of these investigations have indicated that N-linked oligosaccharide side chains play a role in glycoprotein structure and stability, with the structure and stability of different glycoproteins being more or less dependent on the presence of their carbohydrate moieties (Elbein, A. D., Annu. Rev. Biochem., 56:497-534 (1987)).
In experiments aimed at determining whether oligosaccharides play a direct role in promoting protein transport to the cell surface, oligonucleotide-directed mutagenesis has been used to introduce glycosylation sites at novel positions on nascent polypeptides. In some of these cases, the extra oligosaccharides promoted the intracellular transport of mutant proteins that were previously blocked in transport along the secretory pathway (Guan, J. et al, Cell, 42:489-496 (1985); Machamer, C. E. et al, J. Biol. Chem., 263:5948-5954 (1988); and Machamer, C. E. et al, J. Biol. Chem., 263:5955-5960 (1988)). Because the three-dimensional structures of these proteins were not known, it was not possible to predict whether the novel oligosaccharide side chains were attached at positions normally located on the surface or within the interior of the wild-type protein.